Calcium (II) occupies a central role as cofactor of enzymatic proteolysis and as a participant in protein complex formation during blood coagulation. Recent use of trivalent lanthanide ions as substitutes for Ca(II) has facilitated the study of Ca(II)-binding proteins. Studies of the interaction of lanthanide ions with bovine factor X have demonstrated that these ions are competitive inhibitors of Ca(II)-dependent catalysis and are cofactors in metal-dependent protein-complex formation. This strategy has led to the affinity chromatography of the venom protein of Russell's viper using factor X-Sepharose and lanthanide ions. Studies of the substitution of lanthanide ions for Ca(II) in the activation of prothrombin by activated factor X have indicated that lanthanide ions promote formation of thrombin. This phenomena has led to the suggestion that metal ions may play two distinct and separate roles in these systems, promotion of protein complex formation and/or direct participation in catalysis. We propose to extend this previous study to characterize the metal-dependent protein complexes which participate in blood coagulation. The metal binding properties of bovine factors XIa, IX, IXa, VIII, V, and Xa will be studied by the steady state rate dialysis method and by optical and NMR spectroscopy. Possible non-productive metal: protein complex formation in the presence of lanthanide ions will be explored. Affinity chromatography using lanthanide ions to inhibit catalysis and facilitate enzyme-protein substrate interaction where applicable, may provide a method for obtaining quantities of purified clotting proteins for structural and functional characterization and, perhaps, for clinical use.